CN108530561B - Method for extracting high-purity heparan sulfate from heparin production waste - Google Patents
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
Abstract
The invention discloses a method for extracting high-purity heparan sulfate from heparin production waste, and aims to provide the heparan sulfate which is extracted and purified by taking the heparin production waste as a raw material, and the method is low in cost, convenient to operate and easy for industrial amplification; the technical key points are as follows: 1) preparing heparin production waste into solution; 2) adding acid, adjusting pH to 1.9-2.3, adding ethanol, stirring, centrifuging or standing; 3) collecting supernatant, adding alkali solution to adjust pH to 6-8, and adding water; 4) adding an adsorbent into the solution obtained in the step 3), and stirring until the adsorbent adsorbs all mucopolysaccharides in the solution; 5) collecting adsorbent, cleaning with clear water, adding sodium chloride solution for elution, collecting eluent after elution is finished, and adding precipitator for precipitation; 6) collecting precipitate, dissolving in water, and oxidizing with oxidant; 7) adding a precipitator into the oxidized solution for precipitation; 8) collecting the precipitate and drying; belongs to the field of mucopolysaccharide biological pharmacy.
Description
Technical Field
The invention discloses a method for extracting high-purity heparan sulfate, in particular to a method for extracting high-purity heparan sulfate from heparin production waste, belonging to the field of mucopolysaccharide biopharmaceuticals.
Background
Heparin sodium is a commonly used mucopolysaccharide, which in addition to the target product heparin sodium, also usually produces other by-products mucosaccharides such as heparan sulfate, dermatan sulfate, and chondroitin sulfate, wherein chondroitin sulfate in the by-products is easy to remove, and because of the similar properties, dermatan sulfate and heparan sulfate are difficult to separate and purify by using ion exchange chromatography and organic solvent fractional precipitation methods, and generally exist in the form of a mixture without separating the two, or are stored in a warehouse of each production unit, or are directly discarded, thus causing serious waste of resources.
In recent years, with the increasing market of schradett's drugs containing dermatan sulfate, heparan sulfate and heparin as main components, the demand for highly pure compounds of dermatan sulfate and heparan sulfate is more and more urgent, and studies on highly pure heparan sulfate by various researchers have been more and more.
Heparan Sulfate (HS) is a glycosaminoglycan widely present in animal tissue matrices and cell membranes, and its sugar chain is composed of N-acetylglucosamine linked to glucuronic acid or iduronic acid via an α - (1-4) -glycosidic bond to form a disaccharide unit, and is mainly different from Heparin in the degree of sulfated modification of sugar residues, and much less in anticoagulant activity than Heparin.
Fast moving heparin (abbreviated as FM) is named for the component with the faster migration rate of heparin sodium in agarose electrophoresis, compared with the component with the slower migration rate. Fast heparin retains heparin anticoagulant and antithrombotic activities, but the activity is reduced.
Dermatan Sulfate (DS) is a natural glycosaminoglycan widely distributed in animal tissues and composed of repeating disaccharide units, the disaccharide units are acetylgalactosamine Sulfate and iduronic acid, and the disaccharide units are mainly a disaccharide chain structure composed of N-acetylgalactosamine 4-Sulfate and L-iduronic acid. Dermatan sulfate has certain antithrombotic activity and very low anticoagulant activity.
Chinese patent ZL201610010556 discloses a process for extracting heparan sulfate from duodenum, which combines biological enzyme with acid-base treatment method, and the heparan sulfate is obtained by pig duodenum → mincing → enzymolysis → heating → centrifugation → resin adsorption → elution → precipitation → alkali heating → acid treatment → low-power precipitation → oxidation → precipitation → vacuum drying. The patented method is essentially to remove the oil, acidic protein, basic protein and nucleic acid from the duodenum to obtain heparan sulfate, but because other glycosaminoglycans such as dermatan sulfate are also adsorbed by the resin and eluted at direct sodium chloride concentrations, the glycosaminoglycans are mixed with heparan sulfate. Although the subsequent step uses acid-adjusting centrifugation method to remove dermatan sulfate, the method has dermatan sulfate remaining in the supernatant, so that the heparan sulfate is always accompanied with dermatan sulfate, and the purity of the obtained heparan sulfate is not high.
Chinese patent ZL201710220707 discloses a method for preparing heparan sulfate from a heparan sodium crude product extraction waste liquid. In the method, the heparan sulfate is obtained by resin adsorption and elution, and glycosaminoglycan such as dermatan sulfate is mixed, so that the purity of the heparan sulfate is not high.
Chinese patent ZL201410819670 discloses a method for separating heparan sulfate from the lungs of animals, the method selects hydrolytic enzyme to carry out combined enzyme degradation on saline water extract of animal lungs, then adds oxidant and active carbon into enzymatic hydrolysate to carry out oxidation adsorption decoloration, then acetone is adopted to carry out fractional precipitation on the oxidized solution, and the crude heparan sulfate is prepared by dehydration and drying, then sequentially adopting membrane separation and anion exchange chromatography to the aqueous solution of the crude heparan sulfate, separating the heparan sulfate from impurities such as dermatan sulfate, chondroitin sulfate, heparin and the like, then using a 5000-7000Da ultrafiltration membrane to carry out ultrafiltration concentration on the eluent, finally using gel filtration chromatography to desalt and freeze-dry to obtain a heparan sulfate refined product, however, the process flow of the invention is long, complex intermediate process control is needed, the process condition is not easy to be amplified, and the industrialization prospect is great.
Chinese patent ZL200610044491 discloses heparan sulfate from rat tissue and a preparation method thereof, the method comprises the steps of mincing and homogenizing rat tissue, degreasing, hydrolyzing by protease, hydrolyzing by alkali, deproteinizing, centrifuging to obtain clear liquid, dialyzing, precipitating dialyzate by cetyl pyridinium chloride, hydrolyzing by ribonuclease and chondroitinase ABC, and separating by an ion exchange column, and the heparan sulfate obtained by the method has strong anticoagulant activity. However, the higher the purity of heparan sulfate is, the lower the anticoagulation activity is, so the heparan sulfate obtained by the patent is not high in purity.
Chinese patent ZL201510394022 discloses a method for separating heparinoid from heparin byproduct waste protein, which comprises the steps of dissolving the heparin byproduct waste protein, adding an adsorbent, eluting, precipitating with ethanol, oxidizing, precipitating with ethanol, drying to obtain a heparinoid dry product, wherein the heparinoid dry product contains not only heparan sulfate but also mucopolysaccharides such as fast heparin with high anticoagulant activity, which indicates that the purity of the heparan sulfate in the heparinoid dry product is not high.
Chinese patent ZL200910039359 discloses a method for purifying heparan sulfate from heparin byproducts, which comprises the steps of taking the heparin byproducts as raw materials, adding potassium acetate after dissolving, adjusting the pH value of a solution through acetic acid, removing precipitates to obtain a polysaccharide sulfate solution, adding a spot reagent and a saturated sodium hydroxide solution into the polysaccharide sulfate solution, centrifuging, collecting clear liquid, adding the spot reagent and the saturated sodium hydroxide solution into the precipitates, centrifuging, collecting the clear liquid, combining the clear liquid, adding a chromatographic column with anion exchange property, washing away copper ions by using a sodium chloride solution, eluting by using a sodium chloride solution in a linear gradient manner, collecting eluent, evaporating and concentrating the eluent, and precipitating by using ethanol to obtain the high-purity heparan sulfate. The method uses the copper-containing porphyry reagent, the process is not environment-friendly, the subsequent copper removal is needed, the process is complex, and the copper removal effect is unknown.
Chinese patent ZL201210424451 discloses a method for separating and purifying heparin sodium and heparan sulfate from heparin byproducts, which comprises the steps of dissolving raw material heparin byproducts, adding potassium acetate to precipitate and separate the heparin sodium, adding potassium acetate to precipitate after the precipitate is dissolved, collecting the precipitate, and performing multiple fractional precipitation by absolute ethyl alcohol after the precipitate is dissolved to prepare the heparin sodium with higher purity; adding a Banner reagent and a saturated sodium hydroxide solution into the supernatant, centrifuging to obtain a solution containing the heparan sulfate, removing copper ions by using a copper removing agent, adding activated carbon for adsorption, precipitation, decoloring, filtering, adsorbing and eluting by using anion exchange resin, and separating to obtain the high-purity heparan sulfate. The invention uses copper-containing porphyry reagent, the process is not environment-friendly, and the subsequent copper removal is needed, and the process is complex.
Chinese patent ZL200610040707 discloses a method for separating and purifying dermatan sulfate and low molecular heparan sulfate from a sodium heparin by-product, which relates to a method for separating and purifying dermatan sulfate and low molecular heparan sulfate from the sodium heparin by-product, the by-product for producing sodium heparin is taken as a raw material, the raw material is a mixture containing dermatan sulfate and heparan sulfate, the raw material is precipitated by ethanol in a grading way to obtain a crude product of dermatan sulfate; then, nitrite or nitrite ester compounds are used as oxidant to degrade the heparan sulfate contained in the crude product into low molecular heparan sulfate with high solubility, and then high-purity dermatan sulfate and low molecular heparan sulfate are obtained by ethanol fractional precipitation separation. The heparan sulfate obtained by the invention is low molecular weight heparan sulfate obtained by degradation, and is greatly different from the heparan sulfate in the general sense.
Chinese patent ZL01410274835 discloses a method for preparing sulodexide raw material from heparin by-product by ethanol precipitation method, which comprises dissolving heparin by-product, adjusting the solution to high pH with sodium hydroxide, performing low temperature precipitation with appropriate amount of ethanol, and removing precipitate to obtain sulfated polysaccharide solution; regulating the sulfuric acid polysaccharide solution to a low pH value by using hydrochloric acid, performing low-temperature precipitation by using a proper amount of ethanol, and removing the precipitate to obtain a sulfuric acid polysaccharide solution; and then adjusting the pH of the solution to be neutral by using sodium hydroxide, performing room-temperature precipitation by using a proper amount of ethanol, and drying the precipitate to obtain the sulodexide raw material. The sulindac raw material obtained by the invention contains dermatan sulfate and heparin sulfate or heparan sulfate, so that the purity of the heparan sulfate is not high.
In conclusion, no environmental protection process for extracting high-purity heparan sulfate from heparin production waste has been reported so far.
Disclosure of Invention
In view of the above disadvantages, the present invention aims to provide a method for extracting high purity heparan sulfate from heparin production waste, the method takes heparin production waste as raw material for extraction and purification, the cost is low, the operation is convenient, the industrial amplification is easy, and the process method is green and environment-friendly.
In order to solve the above problems, the technical solution provided by the present invention is as follows:
a method for extracting high-purity heparan sulfate from heparin production waste sequentially comprises the following steps:
1) preparing the heparin production waste into a solution with the mass concentration of 5-15%;
2) adding acid into the solution obtained in the step 1), adjusting the pH value to 1.9-2.3, then adding ethanol with the volume 0.1-0.7 times of that of the solution, stirring and centrifuging;
3) collecting supernatant after centrifugation, adding alkaline solution to adjust pH to 6-8, and adding 0-10 times of water to make salinity of the solution less than 2%;
4) adding an adsorbent into the solution obtained in the step 3), and stirring until the adsorbent adsorbs all mucopolysaccharides in the solution;
5) collecting adsorbent, cleaning with clear water, adding sodium chloride solution for eluting for 2-24 hr, collecting eluate, and precipitating with 0.6-1.2 times of precipitant;
6) collecting precipitate, dissolving in water, and oxidizing with oxidant;
7) adding 0.6-1.2 times of heavy precipitator into the oxidized solution for precipitation;
8) collecting the precipitate and drying.
Preferably, in the method for extracting high-purity heparan sulfate from heparin production waste, the acid in the step 2) is one or any combination of hydrochloric acid, trichloroacetic acid and sulfuric acid.
Preferably, in the method for extracting high-purity heparan sulfate from heparin production waste, the adsorbent in the step 4) is ion adsorption resin or ion exchange packing.
Preferably, in the method for extracting high-purity heparan sulfate from heparin production waste, the ion adsorption resin is a strong-base anion adsorption resin, and the resin amount is 5-50ml/g mucopolysaccharide.
Preferably, in the method for extracting high-purity heparan sulfate from heparin production waste, the ion exchange filler is strong anion exchange gel Q Sepharose, and the gel amount is 50-300ml/g mucopolysaccharide.
Preferably, in the above method for extracting high-purity heparan sulfate from heparin production waste, when the adsorbent is strong base anion adsorption resin, the sodium chloride solution in the step 5) has 3-7% salinity, and the addition amount is 0.5-2 times of the volume of the adsorbent.
Preferably, in the above method for extracting high-purity heparan sulfate from heparin production waste, when the adsorbent is strong anion exchange gel Q Sepharose, the sodium chloride in the step 5) is 0.6-1M sodium chloride solution, and the addition amount is 0.5-4 times of the volume of the adsorbent.
Preferably, in the method for extracting high-purity heparan sulfate from heparin production waste, the precipitating agents in the steps 5) and 7) are all one or any combination of ethanol, methanol, acetone and butanone.
Preferably, in the method for extracting high-purity heparan sulfate from heparin production waste, the oxidant in the step 6) is hydrogen peroxide.
Preferably, in the above method for extracting high-purity heparan sulfate from heparin production waste, the alkali solution in step 3) is NaOH solution.
The invention has the beneficial effects that:
1. the technical scheme provided by the invention is that firstly most dermatan sulfate in the waste is removed by an acid-adjusting centrifugal method, and then heparan sulfate is purified by an ion exchange method. The method before the patent almost only uses the ion exchange method to purify the heparan sulfate, but because the separation degree of the dermatan sulfate and the heparan sulfate in the ion exchange method is not enough, the dermatan sulfate is easily adsorbed to the adsorbent in an overload way, so that the heparan sulfate is eluted and is partially eluted at the same time, the dermatan sulfate is mixed in the heparan sulfate, and the purity of the heparan sulfate is finally reduced. The application creatively combines the acid-adjusting centrifugation method and the ion exchange method, namely, after most of dermatan sulfate is removed in a strong acid environment, the heparan sulfate is purified by ion exchange resin or gel, and the purity of the heparan sulfate can be obviously improved.
2. The technical scheme provided by the invention solves the problem that the incomplete structure of the heparan sulfate is easily caused by a method for removing most dermatan sulfate by fractional precipitation of an organic solvent, and the integrity of the heparan sulfate can be furthest reserved by using an acid-adjusting centrifugal method, and the purity of the product is improved.
3. The technical scheme provided by the invention directly separates the high-purity heparan sulfate from the byproducts, effectively solves the problem of financial and material resources required for storing or discarding a large amount of heparin byproducts, changes waste into valuable, recycles the byproducts, saves a large amount of cost, creates value, and finally obtains a product which can be applied to biomedical materials and drug development.
Drawings
FIG. 1 is one of the one-dimensional nuclear magnetic hydrogen spectra of heparan sulfate provided in example 1 of the present invention;
FIG. 2 is the second one-dimensional nuclear magnetic hydrogen spectrum of heparan sulfate provided in example 1 of the present invention.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples.
Example 1:
1) adding water 19kg into 1kg of heparin fine product production waste, and stirring for dissolving to prepare a solution with the concentration of 5%;
2) acid adjustment and centrifugation: adding 1: 1, adjusting the pH value of the solution to 1.9 by HCl, adding 0.1 time of ethanol, stirring and centrifuging;
3) collecting supernatant after centrifugation, adding 30% NaOH solution to adjust pH to 7, and adding 2 times of water to make salinity of the solution less than 2%;
4) adding 50ml of strong base anion adsorption resin (American regison resin) into the solution, stirring and adsorbing for 18 h;
5) separating adsorption residual liquid and resin, cleaning the collected resin with clear water, adding 0.5 time of 3% salinity sodium chloride solution for elution, collecting eluent after 8 hours of elution, and adding 0.8 time of ethanol for precipitation;
6) collecting precipitate, dissolving with 3 times of water, adding hydrogen peroxide for oxidation, and oxidizing for 48 h.
7) Centrifuging the oxidized solution, and adding 0.8 time of ethanol into the centrifuged solution for precipitation;
8) collecting the precipitate, and drying to obtain high-purity heparan sulfate.
Some of the main physicochemical properties of the high purity heparan sulfate obtained in example 1 are as follows:
| item | Standard of merit | Example 1 results |
| anti-FIIa potency | 0-12USPU/mg | 2USPU/mg |
| Molecular weight | 20000-40000Da | 34209Da |
| Purity of | ≥98％ | 99％ |
| DS content | ＜1％ | 0.09％ |
| IV (A + S) content | ≥60％ | 64.39％ |
| IS content | ≤10％ | 7.25％ |
In order to further prove that the heparan sulfate prepared in the example is obtained by performing one-dimensional nuclear magnetic resonance hydrogen spectrum detection on the finished product, referring to fig. 1 and fig. 2, the applicant can prove that the high-purity heparan sulfate obtained in the example has few impurities and high purity.
Example 2:
1) 1kg of heparin fine product production waste is added with 5.67kg of water and stirred to be dissolved to prepare a solution with the concentration of 15 percent;
2) acid adjustment and centrifugation: adding sulfuric acid solution to adjust the pH of the solution to 2.0, adding 0.7 time of ethanol, stirring and centrifuging;
3) collecting the centrifuged supernatant, adding 30% NaOH solution to adjust pH to 6.0, and adding 2.5 times of water to make salinity of the solution less than 2%;
4) adding 500ml of strong basic anion adsorption resin (German Langshen resin) into the solution, stirring and adsorbing for 13 h;
5) separating adsorption residual liquid and resin, cleaning the collected resin with clear water, adding 2 times of 7% salinity sodium chloride solution for elution, collecting eluent after elution for 2 hours, and adding 0.6 times of acetone for precipitation;
6) collecting precipitate, dissolving with 3 times of water, adding hydrogen peroxide for oxidation, and oxidizing for 45 hr.
7) Centrifuging the oxidized solution, and adding 1.2 times of methanol into the centrifuged solution for precipitation;
8) collecting the precipitate, and drying to obtain high-purity heparan sulfate.
Some of the main physicochemical properties of the high purity heparan sulfate obtained in example 2 are as follows:
example 3:
1) adding water 9kg into 1kg of heparin fine product production waste, and stirring for dissolving to prepare a solution with the concentration of 10%;
2) acid adjustment and centrifugation: adding trichloroacetic acid solution to adjust the pH of the solution to 2.3, adding 0.35 time of ethanol, stirring, and standing for 13 h;
3) collecting supernatant after standing, adding 30% NaOH solution to adjust pH to 8, and adding 2 times of water to make salinity of the solution less than 2%;
4) loading the solution onto an ion exchange chromatography column filled with 500ml strong anion exchange gel Q Sepharose;
5) adding 0.5 times of 0.6M sodium chloride solution for elution, collecting eluent at gravity flow rate, and adding 1 time of methanol for precipitation;
6) collecting precipitate, dissolving with 3 times of water, adding hydrogen peroxide for oxidation, and oxidizing for 48 h.
7) Centrifuging the oxidized solution, and adding 0.8 time of acetone into the centrifuged solution for precipitation;
8) collecting the precipitate, and drying to obtain high-purity heparan sulfate.
Some of the main physicochemical properties of the high purity heparan sulfate obtained in example 3 are as follows:
| item | Standard of merit | Example 1 results |
| anti-FIIa potency | 0-12USPU/mg | 3USPU/mg |
| Molecular weight | 20000-40000Da | 33800Da |
| Purity of | ≥98％ | 101％ |
| DS content | ＜1％ | 0.06％ |
| IV (A + S) content | ≥60％ | 64.47％ |
| IS content | ≤10％ | 7.11％ |
Example 4:
1) adding water 19kg into 1kg of heparin fine product production waste, and stirring for dissolving to prepare a solution with the concentration of 5%;
2) acid adjustment and centrifugation: adding hydrochloric acid solution to adjust the pH of the solution to 2.1, adding 0.1 time of ethanol, stirring, and standing for 15 h;
3) collecting supernatant after standing, adding 30% NaOH solution to adjust pH to 8, and adding 2 times of water to make salinity of the solution less than 2%;
4) loading the solution onto an ion exchange chromatography column loaded with 3000ml of strong anion exchange gel Q Sepharose;
5) adding 4 times of 1M sodium chloride solution for elution, collecting eluent at gravity flow rate, and adding 0.8 times of butanone for precipitation;
6) collecting precipitate, dissolving with 3 times of water, adding hydrogen peroxide for oxidation, and oxidizing for 48 h.
7) Centrifuging the oxidized solution, and adding 0.8 time of acetone into the centrifuged solution for precipitation;
8) collecting the precipitate, and drying to obtain high-purity heparan sulfate.
Some of the main physicochemical properties of the high purity heparan sulfate obtained in example 4 are as follows:
| item | Standard of merit | Example 1 results |
| anti-FIIa potency | 0-12USPU/mg | 2USPU/mg |
| Molecular weight | 20000-40000Da | 32800Da |
| Purity of | ≥98％ | 100％ |
| DS content | ＜1％ | 0.06％ |
| IV (A + S) content | ≥60％ | 64.07％ |
| IS content | ≤10％ | 6.92％ |
Example 5:
1) adding water 9kg into 1kg of heparin fine product production waste, and stirring for dissolving to prepare a solution with the concentration of 10%;
2) acid adjustment and centrifugation: adding 1: adjusting the pH of the solution to 2.1 by using 1HCl, adding 0.35 time of ethanol, stirring and centrifuging;
3) collecting supernatant after centrifugation, adding 30% NaOH solution to adjust pH to 6.5, and adding 2.5 times of water to make salinity of the solution less than 2%;
4) adding 300ml of strong basic anion adsorption resin (Rohm and Haas resin) into the solution, stirring and adsorbing for 13 h;
5) separating adsorption residual liquid and resin, cleaning the collected resin with clear water, adding 1.25 times of 3.5% salinity sodium chloride solution for elution, collecting eluent after elution for 24h, and adding 0.8 times of ethanol for precipitation;
6) collecting precipitate, dissolving with 3 times of water, adding hydrogen peroxide for oxidation, and oxidizing for 48 h.
7) Centrifuging the oxidized solution, and adding 1.2 times of methanol into the centrifuged solution for precipitation;
8) collecting the precipitate, and drying to obtain high-purity heparan sulfate.
Some of the main physicochemical properties of the high purity heparan sulfate obtained in example 5 are as follows:
| item | Standard of merit | Example 1 results |
| anti-FIIa potency | 0-12USPU/mg | 4USPU/mg |
| Molecular weight | 20000-40000Da | 32468Da |
| Purity of | ≥98％ | 99％ |
| DS content | ＜1％ | 0.09％ |
| IV (A + S) content | ≥60％ | 63.92％ |
| IS content | ≤10％ | 7.04％ |
Example 6:
1) 1kg of heparin fine product production waste is added with 5.67kg of water and stirred to be dissolved to prepare a solution with the concentration of 15 percent;
2) acid adjustment and centrifugation: adding hydrochloric acid solution to adjust the pH of the solution to 2.3, adding 0.7 time of ethanol, stirring, and standing for 15 h;
3) collecting supernatant after standing, adding 30% NaOH solution to adjust pH to 8, and adding 2 times of water to make salinity of the solution less than 2%;
4) loading the solution onto an ion exchange chromatography column filled with 1750ml of strong anion exchange gel Q Sepharose;
5) adding 2.3 times of 0.8M sodium chloride solution for elution, collecting eluent at gravity flow rate, and adding 1 time of methanol for precipitation;
6) collecting precipitate, dissolving with 3 times of water, adding hydrogen peroxide for oxidation, and oxidizing for 48 h.
7) Centrifuging the oxidized solution, and adding 0.8 time of acetone into the centrifuged solution for precipitation;
8) collecting the precipitate, and drying to obtain high-purity heparan sulfate.
Some of the main physicochemical properties of the high purity heparan sulfate obtained in example 6 are as follows:
| item | Standard of merit | Example 1 results |
| anti-FIIa potency | 0-12USPU/mg | 2USPU/mg |
| Molecular weight | 20000-40000Da | 32956Da |
| Purity of | ≥98％ | 100％ |
| DS content | ＜1％ | 0.07％ |
| IV (A + S) content | ≥60％ | 64.79％ |
| IS content | ≤10％ | 6.68％ |
Finally, it should be noted that the above-mentioned embodiments are only specific embodiments of the present invention, and obviously, the present invention is not limited to the above-mentioned embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (6)
1. A method for extracting high-purity heparan sulfate from heparin production waste is characterized by sequentially comprising the following steps:
1) preparing the heparin production waste into a solution with the mass concentration of 5-15%;
2) adding acid into the solution obtained in the step 1), adjusting the pH value to 1.9-2.3, then adding ethanol with the volume of 0.1-0.7 times of that of the solution, stirring, centrifuging or standing for layering;
3) collecting supernatant after centrifugation or standing for layering, adding alkaline solution to adjust pH to 6-8, and adding 0-10 times of water to make salinity of the solution less than 2%;
4) adding an adsorbent into the solution obtained in the step 3), and stirring until the adsorbent adsorbs all mucopolysaccharides in the solution;
5) collecting adsorbent, cleaning with clear water, adding sodium chloride solution for eluting for 2-24 hr, collecting eluate, and precipitating with 0.6-1.2 times of precipitant;
6) collecting precipitate, dissolving in water, and oxidizing with oxidant;
7) adding 0.6-1.2 times of heavy precipitator into the oxidized solution for precipitation;
8) collecting the precipitate and drying;
the acid in the step 2) is one or any combination of hydrochloric acid, trichloroacetic acid and sulfuric acid;
the adsorbent in the step 4) is ion adsorption resin or ion exchange filler;
the ion adsorption resin is strong-base anion adsorption resin, and the resin amount is 5-50mL/g mucopolysaccharide;
the ion exchange filler is strong anion exchange gel Q Sepharose, and the gel amount is 50-300ml/g mucopolysaccharide.
2. The method of claim 1, wherein the sodium chloride solution in step 5) is: the addition amount of sodium chloride solution with the salinity of 3-7 percent is 0.5-2 times of the volume of the adsorbent.
3. The method of claim 1, wherein the sodium chloride solution in step 5) is: 0.6-1M sodium chloride solution, the addition amount is 0.5-4 times of the volume of the adsorbent.
4. The method of claim 1, wherein the precipitating agent in step 5) and step 7) is one or any combination of ethanol, methanol, acetone, and butanone.
5. The method for extracting high-purity heparan sulfate from heparin production waste according to claim 1, wherein the oxidant in step 6) is hydrogen peroxide.
6. The method of claim 1, wherein the alkali solution in step 3) is NaOH solution.
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| CN110540607A (en) * | 2019-09-24 | 2019-12-06 | 潢川县鹏升畜产品有限公司 | treatment method of heparin sodium extraction waste liquid |
| CN111154012A (en) * | 2019-12-31 | 2020-05-15 | 湖北亿诺瑞生物制药有限公司 | Preparation method of ultra-high purity heparan sulfate |
| CN114940718A (en) * | 2022-06-14 | 2022-08-26 | 湖北亿诺瑞生物制药有限公司 | Method for extracting chondroitin sulfate from heparin leftovers |
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| CN105037584A (en) * | 2015-07-02 | 2015-11-11 | 杭州惠顺生物科技有限公司 | Method for separating heparitin from heparin by-product waste protein |
| CN105504098A (en) * | 2016-01-08 | 2016-04-20 | 东营天东制药有限公司 | Technology for extracting heparin sulfate from duodena |
| CN106905446A (en) * | 2017-04-06 | 2017-06-30 | 山东绅联药业有限公司 | A kind of sheep heparin sodium crude extracts the method that waste liquid prepares Heparan sulfate |
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| CN105037584A (en) * | 2015-07-02 | 2015-11-11 | 杭州惠顺生物科技有限公司 | Method for separating heparitin from heparin by-product waste protein |
| CN105504098A (en) * | 2016-01-08 | 2016-04-20 | 东营天东制药有限公司 | Technology for extracting heparin sulfate from duodena |
| CN106905446A (en) * | 2017-04-06 | 2017-06-30 | 山东绅联药业有限公司 | A kind of sheep heparin sodium crude extracts the method that waste liquid prepares Heparan sulfate |
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